Method for the diagnosis of Helicobacter pylori infection, and a diagnostic kit for performing the method

ABSTRACT

The method for the diagnosis of  Helicobacter pylori  infection by the oral administration of defined amounts of  13 C-labeled urea and examination for  13 C content of blood samples removed at a defined time is effected by  
     a) removing from 0.1 to 0.6 ml of capillary blood from the finger or ear lob of a patient or venous blood of a patient, in both cases with an empty stomach before the beginning of the test;  
     b) administering an exact amount of from 10 to 50 mg of  13 C-urea in aqueous solution with a pH value of 2 to 4 to the patient;  
     c) again removing capillary or venous blood exactly after 10 to 15 min from the administration; and  
     d) determining the  13 C content of the blood samples by isotope ratio mass spectrometry (IRMS), and deducing the presence of  Helicobacter pylori  from the increase of the  13 C values.

[0001] The present invention relates to a method for the diagnosis ofHelicobacter pylori infection by the oral administration of definedamounts of ¹³C-labeled urea and examination for ¹³C content of bloodsamples removed at a defined time.

[0002] The previously usual and mostly performed method for thediagnosis of Helicobacter pylori infection is the ¹³C-urea respirationtest. This method has been described in detail in EP-A-0 253 927. Thistest has the disadvantage, that it cannot be applied for children below3 years and adults suffering from breathinsufficiency like asthma.

[0003] Rex Moulton-Barrett et al., The American Journal ofGastroenterology, Vol. 88, 1993, pages 369 to 374, describe a method inwhich ¹³C-labeled hydrogencarbonate is determined in the serum upon oraladministration of ¹³C-labeled urea. In this test, 5 mg/kg of ¹³C-labeledurea was administered to the patient, and blood samples of 3 ml eachwere taken and examined after 15, 30, 60, 90, 120 and 180 min. Thismethod has been further examined and described by Mark J. Kim et al. inGastroenterology 1997, 113: 31-37, W. D. Chey et al. in The AmericanJournal of Gastroenterology, Vol. 94, 1999, pages 1522 to 1524, Alan F.Cuttler et al. in The American Journal of Gastroenterology, Vol. 94,1999, pages 959 to 961, and has resulted in the introduction of a testby the company Metabolic Solutions Inc., Nashua, N.H. In this test,without determination of the zero value and upon administration of ahigh-fat test meal “Ensure”, 125 mg of ¹³C-urea is administered, and the¹³C content of 3 ml of blood is determined after 30 min. However, due tothe low accuracy and precision and due to the very high price of thenecessary amount of ¹³C-urea and the relatively high test quantity ofblood, this test was not successful economically, so that therespiration test remained the mostly used test method despite of all itsdisadvantages.

[0004] It has been the object of the invention to provide a method forthe diagnosis of Helicobacter pylori infection with as low as possiblean amount of ¹³C-urea and as low as possible a blood quantity for theexamination of the ¹³C content, which method establishes the diagnosisof Helicobacter pylori infection more simply, more inexpensively, moreaccurately and more quickly.

[0005] This object is now achieved by removing from 0.1 to 0.6 ml ofcapillary blood from the finger or ear lob of a patient or venous blood,in both cases with an empty stomach before the beginning of the test,administering an exact amount of from 10 to 50 mg of ¹³C-urea in aqueoussolution with a pH value of 2 to 4 to the patient, again removingcapillary or venous blood exactly after 10 to 15 min from theadministration, and determining the ¹³C content of the blood samples byisotope ratio mass spectrometry (IRMS), and deducing the presence ofHelicobacter pylori from the increase of the ¹³C values.

[0006] Even when 0.6 ml of capillary blood and 50 mg of ¹³C-urea isused, this method is clearly superior to the previous method, all themore so since the accuracy of the method is enormously increased bydetermining the starting value, and the method is significantlyabbreviated for the patient by the removal of a second blood samplealready after 10 to 15 min. In elder patients, the recovery of capillaryblood sometimes involves difficulties. In such cases, the method canalso be performed with the same small amount of venous blood.

[0007] Of critical importance in the method according to the inventionis the omission of the high-fat test meal. Instead, the labeled urea isadministered in an aqueous solution with a pH value of from 2 to 4. Thiscan be done, for example, either by adjusting the aqueous solution ofthe labeled urea by means of a non-volatile, pharmacologicallyacceptable organic acid to a pH value of from 2 to 4, or by adding apack of a solid pharmacologically acceptable organic acid to the freshlyprepared solution. Citric acid has been found particularly suitable.However, in principle, other organic acids of that kind, such asascorbic acid, are also capable of adjusting the desired pH value. It isalso possible to achieve this low pH value by the use of orange juice,grapefruit juice or sour apple juice.

[0008] The determination of the ¹³C content in the blood samples can beeffected, for example, by adding a strong non-volatile acid, such asphosphoric acid, which is capable to release the carbon dioxide in agaseous form from the blood sample, so that it can be measured with anIRMS device.

[0009] However, it is also possible to recover serum from the bloodsamples, to remove high molecular weight components from the serumsample by means of suitable filters, and to determine the ¹³C content inthe remaining liquid by a preliminary elemental analysis followed byisotope ratio mass spectrometry.

[0010] By comparative examinations, it was established that thedetection of a Helicobacter pylori infection in a respiratory test ispositive when the value of ¹³C in the respiratory air is about 4% abovethe starting value. In contrast, the method according to the inventionis capable of detecting Helicobacter infections when the difference ofthe ¹³C content is as low as 2.0%. It is obvious that the substantiallylower amount of the expensive ¹³C-urea, on the one hand, and thequickening of the time of the second sampling from 30 min to 10 min areof significant importance to both the patients and the physicians. Inaddition, the removal of a maximum of 0.6 ml of capillary or venousblood is significantly simpler and more convenient than the removal of 3ml of venous blood in the test of the Metabolic Solutions Inc. Ascompared to this test, above all, the significantly higher accuracy andprecision is of critical importance since the starting value isdetermined for each patient rather than using the average values, whichare accompanied by considerable variations and influenced by thedifferent foods taken up by the subjects.

[0011] The diagnostic kit according to the invention for performing themethod preferably consists of an acidic aqueous solution having a pHvalue of from 2 to 4 and containing exactly from 10 to 50 mg of¹³C-urea, a patient instruction sheet, two sample vessels for receivingthe blood samples, and optionally a blood-sampling device. It isdecisive that the kit contains an exaxt amount of ¹³C-urea. A kit forchildren may contain less than for adults in the over all range of 10 to50 mg ¹³C-urea. Also the time difference between the two removals ofblood should be exactly measured. Different kits, however, may be run inthe over all time range of 10 to 15 min.

[0012] Another preferred embodiment comprises instead of the readyacidic solution a container for urea with exactly from 10 to 50 mg of¹³C-urea and a pack of a solid pharmacologically acceptable organicacid, such as citric acid. Normally, 2 g of citric acid in 200 ml ofwater or 200 ml of orange juice, grapefruit juice or sour apple juice issuitable for dissolving from 30 to 50 mg of ¹³C-urea. Especially forchildren, the amount of ¹³C-urea can be further decreased down to 10 mg.

[0013] The blood samples preferably are taken up in commerciallyavailable sample vessels for receiving blood samples, for example,Vacutainer®. Preferably, they may already contain the necessary amountof concentrated phosphoric acid, so that the CO₂ is immediately releasedfrom the blood sample. In principle, however, it is also possible to adda strong non-volatile acid later to the sample vessel for receiving theblood sample.

[0014] The determination of the ¹³C content is then effected by IRMSdirectly from the gas phase. An increase of the ¹³C content of as low as2% after 10 min indicates infection with Helicobacter pylori.

[0015] Alternatively, serum may also be withdrawn from the blood sampleafter a short settling period. All macromolecules and especially thelipids can be removed from this serum sample by ultrafiltration. Theremaining liquid is then first subjected to elemental analysis bycombustion, and the CO₂ released thereby is again examined by IRMS forthe isotope ratio ¹³C/¹²C. In this test method, infection withHelicobacter pylori can be detected at an increase of the ¹³C content ofas low as from 1 to 1.5%.

[0016] The higher sensitivities and accuracies of the test methodachieved according to the invention can be explained afterwards by thefact that the ¹³C content of the labeled urea is to a lesser extentdiluted with other carbon sources. This dilution effect is strongest inthe respiratory air test and least in the examination of serum samplesfrom which macromolecular molecules are removed by ultrafiltration.Nevertheless, the method in which CO₂ is released from the blood sampleby a strong non-volatile acid and determined in the gas phase is alreadyexcellently suitable for achieving the object of the present invention.

[0017] When the method according to the invention is performed inpractice, the blood tests are executed by the physician before and from10 to 15 min after administration of the ¹³C-urea. The Vacutainers® canbe collected and sent to a central laboratory where the determination ofthe ¹³C contents is effected. From these results fed-back to thephysician, he can diagnose whether or not there is an infection withHelicobacter pylori.

[0018] In principle, all highly sensitive isotope mass spectrometersavailable on the market can be used for the IRMS determination; however,due to their high price, they can be set up only in centrallaboratories. Already for clinics, investment in such devices cannot beexpected. In contrast, the collecting and analyzing of samples incentral laboratories does not involve any problems today in terms oflogistics and prices, and the same applies to the back transmission ofanalytical data to the submitting physician or the submitting clinic.

[0019] The same is true of the determination method in the serum fromwhich high molecular weight components have been removed byultrafiltration. The preliminary elemental analysis by combustion can beperformed in central laboratories without problems. The recovery ofserum from capillary or venous blood and ultrafiltration can beperformed without problems at least in clinics.

[0020] For the actual determination of the ¹³C content, 20 μl of serumis sufficient and will yield telling results already for an increase offrom 1 to 1.5% of ¹³C.

1. A method for the diagnosis of Helicobacter pylori infection by the oral administration of defined amounts of ¹³C-labeled urea and examination for ¹³C content of blood samples removed at a defined time, characterized in that a) from 0.1 to 0.6 ml of capillary blood is removed from the finger or ear lob of a patient or venous blood of a patient, in both cases with an empty stomach before the beginning of the test; b) an exact amount of from 10 to 50 mg of ¹³C-urea in aqueous solution with a pH value of 2 to 4 is administered to the patient; c) capillary or venous blood is again removed exactly after 10 to 15 min from the administration; and d) the ¹³C content of the blood samples is determined by isotope ratio mass spectrometry (IRMS), and the presence of Helicobacter pylori is deduced from the increase of the ¹³C values.
 2. The method according to claim 1, characterized in that a strong non-volatile acid which releases CO₂ in a gaseous form is added to said samples, and the ¹³C contents are determined in the gas phase.
 3. The method according to claim 1, characterized in that serum is recovered from the blood samples, high molecular weight components are removed from the serum by filters, and the ¹³C content is determined in the remaining liquid by a preliminary elemental analysis followed by mass spectrometry.
 4. A diagnostic kit for performing the method according to any of claims 1 to 3, consisting of 1) either an acidic aqueous solution having a pH value of from 2 to 4 and containing exactly from 10 to 50 mg of ¹³C-urea, or 1a) a urea container with exactly from 10 to 50 mg of ¹³C-urea; 1b) a pack of a solid pharmacologically acceptable organic acid; 2) a patient instruction sheet; 3) two sample vessels for receiving the blood samples which either 3a) already contain a strong non-volatile acid; or 3b) enable the addition of such an acid; and optionally 4) a blood-sampling device.
 5. A diagnostic kit according to claim 4, characterized in that said organic acid is citric acid. 